TW202207932A - Therapeutic agent for RNA viral infection obtained by combining pyrazine derivative and thiopurine derivative - Google Patents

Abstract

The present invention addresses the problem of providing a therapeutic agent for RNA viral infection that includes a novel combination of a pyrazine derivative and a specific compound, the novel combination exhibiting an effect against the RNA virus. The present invention also addresses the problem of providing a therapeutic agent for RNA viral infection that includes a combination of a pyrazine derivative and a specific compound, the combination being capable of simultaneously enhancing anti-virus activities against multiple RNA viruses. The present invention provides a therapeutic agent for RNA viral infection obtained by combining a pyrazine derivative or a salt thereof and a thiopurine derivative.

Description

Therapeutic agent for RNA virus infection composed of a combination of pyridine derivative and thiopurine derivative

衍生物或其鹽與硫嘌呤衍生物而構成的RNA病毒感染症治療劑。The present invention relates to combination pyridine

A therapeutic agent for RNA virus infection comprising a derivative or a salt thereof and a thiopurine derivative.

衍生物(非專利文獻1)。RNA viruses such as influenza virus and Ebola virus cause various infectious diseases, and countermeasures are required. On the other hand, pyridoxine such as favipiravir (hereinafter, also referred to as T-705) is known as a compound having a wide range of antiviral activity against many RNA viruses.

Derivatives (Non-Patent Document 1).

衍生物的活性，則作為醫藥更為有用。迄今為止，已有報告：藉由組合法匹拉韋與神經胺糖酸酶(neuraminidase)抑制劑，而對流感病毒顯示相乘的效果(專利文獻1)。又，亦已有報告：藉由組合法匹拉韋與吉西他濱(gemcitabine)或奥巴克拉(obatoclax)，而對伊波拉病毒顯示相乘的效果(專利文獻2)。If these pyridines can be enhanced

The activity of derivatives is more useful as medicine. Heretofore, it has been reported that a combination of favipiravir and a neuraminidase inhibitor exhibits a synergistic effect on influenza virus (Patent Document 1). In addition, it has also been reported that by combining favipiravir with gemcitabine or obatoclax, a synergistic effect is shown on Ebola virus (Patent Document 2).

However, RNA viruses have evolved to acquire drug resistance. For example, influenza viruses that are resistant to neuraminidase inhibitors are known (Non-Patent Document 2). In order to counteract such resistance acquisition, new combinations of compounds showing effects on RNA viruses are constantly being sought.

衍生物與特定之化合物的組合。As also mentioned above, it is characteristic of favipiravir that it exhibits a broad range of effects against many RNA viruses. However, pyridoxine that can simultaneously enhance antiviral activity against multiple RNA viruses is not known.

Combinations of derivatives and specific compounds.

6-methylmercaptopurine riboside, which is a thiopurine derivative, is known to have a certain antiviral activity (Non-Patent Document 3). However, it is not known at all what effect it exhibits in cells when used in combination with favipiravir. [Prior Art Literature] [Patent Literature]

[Patent Document 1] International Publication No. 08/099874 Pamphlet [Patent Document 2] International Publication No. 2007/202789 Pamphlet [Non-patent literature]

[Non-Patent Document 1] Proc Jpn Acad Ser B Phys Biol Sci. 93, 449-463. [Non-Patent Literature 2] CDC Health Advisory: CDC issues interim recommendations for the use of influenza antivirals in the setting of oseltamivir Resistance among circulating influenza A (H1N1) viruses, 2008–09 Season. [Non-Patent Document 3] PLoS One. October 2011, Volume 6, Issue 10, e26697

[The problem to be solved by the invention]

衍生物與特定之化合物之新組合的RNA病毒感染症治療劑，其對RNA病毒顯示效果。又本發明之另外的課題為提供一種包含吡

衍生物與特定之化合物之組合的RNA病毒感染症治療劑，其可同時增強對複數之RNA病毒的抗病毒活性。 [用以解決課題之手段]The subject of the present invention is to provide a

A novel combination of a derivative and a specific compound is a therapeutic agent for RNA virus infection, which shows an effect on RNA virus. Another subject of the present invention is to provide a pyridine-containing

A therapeutic agent for RNA virus infection, which is a combination of a derivative and a specific compound, which can simultaneously enhance the antiviral activity against a plurality of RNA viruses. [means to solve the problem]

衍生物或其鹽

(式中，R¹ 及R² 係相同或相異而表示氫原子或鹵素原子；R³ 表示氫原子或胺基保護基)、與 6-甲基巰基嘌呤核糖苷等之硫嘌呤衍生物組合而使用，則對RNA病毒的抗病毒活性會增強，而此增強作用之至少一部分係由於細胞內的吡

衍生物核糖三磷酸體的量的增加所致，遂而完成本發明。Under such circumstances, the inventors of the present invention, as a result of intensive examination, found that if the pyridine represented by the general formula [1]

Derivatives or their salts

(in the formula, R ¹ and R ² are the same or different and represent a hydrogen atom or a halogen atom; R ³ represents a hydrogen atom or an amine protecting group), combined with thiopurine derivatives such as 6-methylmercaptopurine riboside and use, the antiviral activity against RNA viruses is enhanced, and at least part of this enhancement is due to intracellular pyridoxine

The present invention was completed due to the increase in the amount of the derivative ribose triphosphate.

衍生物或其鹽

(式中，R¹ 及R² 係相同或相異而表示氫原子或鹵素原子；R³ 表示氫原子或胺基保護基)、與 通式[2]所表示的硫嘌呤衍生物

(式中，R⁴ 及R⁵ 係相同或相異而表示氫原子或羥基保護基；R⁶ 係表示氫原子、可經保護的一磷酸基、可經保護的二磷酸基或可經保護的三磷酸基；R⁷ 係表示氫原子或可經保護的胺基；R⁸ 係表示氫原子、C_1-6 烷基或通式[3]所表示的基

(式中，R⁹ 係表示氫原子、C_1-6 烷基或羧基；R¹⁰ 係表示氫原子、C_1-6 烷基、苄基或p-硝基苄基)；X係表示氧原子、硫原子、碳原子或可經保護的亞胺基)而構成。 [2]如[1]記載之治療劑，其中R¹ 為氫原子，R² 為氟原子或氫原子，及R³ 為氫原子。 [3]如[1]或[2]記載之治療劑，其中R¹ 為氫原子，R² 為氟原子，及R³ 為氫原子。 [4]如[1]～[3]中任一項記載之治療劑，其中R⁴ 、R⁵ 、R⁶ 及R⁷ 為氫原子，R⁸ 為甲基，及X氧原子。That is, the present invention provides the following. [1] A therapeutic agent for RNA virus infection, which is a combination of pyridoxine represented by the general formula [1]

Derivatives or their salts

(in the formula, R ¹ and R ² are the same or different and represent a hydrogen atom or a halogen atom; R ³ represents a hydrogen atom or an amino protecting group), and the thiopurine derivative represented by the general formula [2]

(in the formula, R ⁴ and R ⁵ are the same or different and represent a hydrogen atom or a hydroxyl protective group; R ⁶ represents a hydrogen atom, a monophosphate group that can be protected, a diphosphate group that can be protected or a protective group that can be protected Triphosphoric acid group; R ⁷ represents a hydrogen atom or a protectable amine group; R ⁸ represents a hydrogen atom, a C _1-6 alkyl group or a group represented by the general formula [3]

(in the formula, R ⁹ represents a hydrogen atom, a C _1-6 alkyl group or a carboxyl group; R ¹⁰ represents a hydrogen atom, a C _1-6 alkyl group, a benzyl group or a p-nitrobenzyl group); X represents an oxygen atom , sulfur atom, carbon atom or protected imine group). [2] The therapeutic agent according to [1], wherein R ¹ is a hydrogen atom, R ² is a fluorine atom or a hydrogen atom, and R ³ is a hydrogen atom. [3] The therapeutic agent according to [1] or [2], wherein R ¹ is a hydrogen atom, R ² is a fluorine atom, and R ³ is a hydrogen atom. [4] The therapeutic agent according to any one of [1] to [3], wherein R ⁴ , R ⁵ , R ⁶ and R ⁷ are hydrogen atoms, R ⁸ is a methyl group, and X is an oxygen atom.

衍生物或其鹽

(式中，R¹ 及R² 係相同或相異而表示氫原子或鹵素原子；R³ 表示氫原子或胺基保護基)、與 通式[2]所表示的硫嘌呤衍生物

(式中，R⁴ 及R⁵ 係相同或相異而表示氫原子或羥基保護基；R⁶ 係表示氫原子、可經保護的一磷酸基、可經保護的二磷酸基或可經保護的三磷酸基；R⁷ 係表示氫原子或可經保護的胺基；R⁸ 係表示氫原子、C_1-6 烷基或通式[3]所表示的基

(式中，R⁹ 係表示氫原子、C_1-6 烷基或羧基；R¹⁰ 係表示氫原子、C_1-6 烷基、苄基或p-硝基苄基)；X係表示氧原子、硫原子、碳原子或可經保護的亞胺基)。 [B]一種通式[1]所表示的吡

衍生物或其鹽與通式[2]所表示的硫嘌呤衍生物之用途，其係用於製造RNA病毒感染症治療藥， 其中通式[1]為

(式中，R¹ 及R² 係相同或相異而表示氫原子或鹵素原子；R³ 表示氫原子或胺基保護基) 通式[2]為

(式中，R⁴ 及R⁵ 係相同或相異而表示氫原子或羥基保護基；R⁶ 係表示氫原子、可經保護的一磷酸基、可經保護的二磷酸基或可經保護的三磷酸基；R⁷ 係表示氫原子或可經保護的胺基；R⁸ 係表示氫原子、C_1-6 烷基或通式[3]所表示的基

(式中，R⁹ 係表示氫原子、C_1-6 烷基或羧基；R¹⁰ 係表示氫原子、C_1-6 烷基、苄基或p-硝基苄基)；X係表示氧原子、硫原子、碳原子或可經保護的亞胺基)。 [C]一種通式[1]所表示的吡

衍生物或其鹽與通式[2]所表示的硫嘌呤衍生物之組合，其係用以於RNA病毒感染症之治療中使用， 其中通式[1]為

(式中，R¹ 及R² 係相同或相異而表示氫原子或鹵素原子；R³ 表示氫原子或胺基保護基) 通式[2]為

(式中，R⁴ 及R⁵ 係相同或相異而表示氫原子或羥基保護基；R⁶ 係表示氫原子、可經保護的一磷酸基、可經保護的二磷酸基或可經保護的三磷酸基；R⁷ 係表示氫原子或可經保護的胺基；R⁸ 係表示氫原子、C_1-6 烷基或通式[3]所表示的基

(式中，R⁹ 係表示氫原子、C_1-6 烷基或羧基；R¹⁰ 係表示氫原子、C_1-6 烷基、苄基或p-硝基苄基)；X係表示氧原子、硫原子、碳原子或可經保護的亞胺基)。 [發明之效果]The present invention further provides the following. [A] A method for treating an RNA virus infection, comprising administering to a subject: pyridine represented by the general formula [1]

Derivatives or their salts

(in the formula, R ¹ and R ² are the same or different and represent a hydrogen atom or a halogen atom; R ³ represents a hydrogen atom or an amino protecting group), and the thiopurine derivative represented by the general formula [2]

(in the formula, R ⁴ and R ⁵ are the same or different and represent a hydrogen atom or a hydroxyl protective group; R ⁶ represents a hydrogen atom, a monophosphate group that can be protected, a diphosphate group that can be protected or a protective group that can be protected Triphosphoric acid group; R ⁷ represents a hydrogen atom or a protectable amine group; R ⁸ represents a hydrogen atom, a C _1-6 alkyl group or a group represented by the general formula [3]

(in the formula, R ⁹ represents a hydrogen atom, a C _1-6 alkyl group or a carboxyl group; R ¹⁰ represents a hydrogen atom, a C _1-6 alkyl group, a benzyl group or a p-nitrobenzyl group); X represents an oxygen atom , a sulfur atom, a carbon atom or a protectable imino group). [B] A pyridine represented by the general formula [1]

Use of a derivative or a salt thereof and a thiopurine derivative represented by the general formula [2] for the manufacture of a therapeutic drug for RNA virus infection, wherein the general formula [1] is

(in the formula, R ¹ and R ² are the same or different and represent a hydrogen atom or a halogen atom; R ³ represents a hydrogen atom or an amino protecting group) The general formula [2] is

(in the formula, R ⁴ and R ⁵ are the same or different and represent a hydrogen atom or a hydroxyl protective group; R ⁶ represents a hydrogen atom, a monophosphate group that can be protected, a diphosphate group that can be protected or a protective group that can be protected Triphosphoric acid group; R ⁷ represents a hydrogen atom or a protectable amine group; R ⁸ represents a hydrogen atom, a C _1-6 alkyl group or a group represented by the general formula [3]

(in the formula, R ⁹ represents a hydrogen atom, a C _1-6 alkyl group or a carboxyl group; R ¹⁰ represents a hydrogen atom, a C _1-6 alkyl group, a benzyl group or a p-nitrobenzyl group); X represents an oxygen atom , a sulfur atom, a carbon atom or a protectable imino group). [C] a pyridine represented by the general formula [1]

A combination of a derivative or a salt thereof and a thiopurine derivative represented by the general formula [2], which is used in the treatment of RNA virus infections, wherein the general formula [1] is

(in the formula, R ¹ and R ² are the same or different and represent a hydrogen atom or a halogen atom; R ³ represents a hydrogen atom or an amino protecting group) The general formula [2] is

(in the formula, R ⁴ and R ⁵ are the same or different and represent a hydrogen atom or a hydroxyl protective group; R ⁶ represents a hydrogen atom, a monophosphate group that can be protected, a diphosphate group that can be protected or a protective group that can be protected Triphosphoric acid group; R ⁷ represents a hydrogen atom or a protectable amine group; R ⁸ represents a hydrogen atom, a C _1-6 alkyl group or a group represented by the general formula [3]

(in the formula, R ⁹ represents a hydrogen atom, a C _1-6 alkyl group or a carboxyl group; R ¹⁰ represents a hydrogen atom, a C _1-6 alkyl group, a benzyl group or a p-nitrobenzyl group); X represents an oxygen atom , a sulfur atom, a carbon atom or a protectable imino group). [Effect of invention]

衍生物或其鹽、與硫嘌呤衍生物而成的RNA病毒感染症治療劑，係有用於RNA病毒感染症之治療或預防等之處置。combination pyridine

The therapeutic agent for RNA virus infection comprising the derivative or its salt and the thiopurine derivative is used for the treatment or prevention of RNA virus infection.

[Form for carrying out the invention]

Hereinafter, the present invention will be described in detail. The halogen atom means a fluorine atom, a chlorine atom, a bromine atom and an iodine atom. C _1-6 alkyl means methyl, ethyl, propyl, isopropyl, butyl, tertiary butyl, isobutyl, tertiary butyl, pentyl, isopentyl, 2-methylbutyl linear or branched C _1-6 alkyl groups such as radicals, 2-pentyl, 3-pentyl and hexyl.

The amino protecting group includes all the groups that can be used as the amino protecting group. For example, W. Greene et al., Protective Groups in Organic Synthesis 5th Edition , pp. 895-1193, 2014, based on John Wiley & Sons, INC.

Specifically, for example, an acyl group, an alkoxycarbonyl group, an arylalkoxycarbonyl group, an aryloxycarbonyl group, an arylalkyl group, an alkoxyalkyl group, an arylalkoxyalkyl group, and an arylthio group can be mentioned. , alkylsulfonyl, arylsulfonyl, dialkylaminoalkylene, arylalkylene, nitrogen-containing heterocyclic alkylene, cycloalkylene, diarylphosphory , diarylalkyl phosphoric acid groups, oxygen-containing heterocyclic alkyl groups and substituted silyl groups, etc.

For the imino protecting group, it includes all the groups that can be used as the protecting group of the imino group, for example, W. Greene et al., Protective Groups in Organic Synthesis (Protective Groups in Organic Synthesis) ) 5th Edition, pp. 895-1115, 2014, based on John Wiley & Sons, INC. Specifically, aryl C _1-6 alkyl, C _1-6 alkoxy C _1-6 alkyl, acyl group, C _1-6 alkoxycarbonyl, aryl C _1-6 alkoxycarbonyl, Aryloxycarbonyl, C _1-6 alkylsulfonyl, arylsulfonyl or silyl group.

The hydroxyl protecting group includes all groups that can be generally used as a protecting group for a hydroxyl group, for example, W. Greene et al., Protective Groups in Organic Synthesis 5th Edition , pp. 17-471, 2014, based on John Wiley & Sons, INC.

Specifically, for example, C _1-6 alkyl, aryl C _1-6 alkyl, C _1-6 alkoxy C _1-6 alkyl, acyl group, C _1-6 alkoxycarbonyl, aryl C _1-6 alkoxycarbonyl, C _1-6 alkylsulfonyl, arylsulfonyl, silyl, tetrahydrofuranyl or tetrahydropyranyl.

C _1-6 alkyl means straight chain of methyl, ethyl, propyl, isopropyl, butyl, tertiary butyl, isobutyl, tertiary butyl, pentyl, isopentyl and hexyl, etc. C _1-6 alkyl in the shape or branched chain. The ar-C _1-6 alkyl group means an ar-C _1-6 alkyl group such as benzyl, diphenylmethyl, trityl, phenethyl, and naphthylmethyl. C _1-6 alkoxy C _1-6 alkyl means C _1-6 alkoxy C _1-6 alkyl such as methoxymethyl and 1-ethoxyethyl. Acyl group means methanoyl group, C _2-6 alkanoyl group, aryl group or heterocyclic carbonyl group. C _1-6 alkoxycarbonyl means one of methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, tertiary butoxycarbonyl and 1,1-dimethylpropoxycarbonyl, etc. Linear or branched C _1-6 alkoxycarbonyl. The ar-C _1-6 alkoxycarbonyl group means an ar-C _1-6 alkoxycarbonyl group such as benzyloxycarbonyl and phenethyloxycarbonyl. C _1-6 alkylsulfonyl group means C _1-6 alkylsulfonyl group such as methylsulfonyl group, ethylsulfonyl group and propylsulfonyl group. Arylsulfonyl means benzenesulfonyl, p-toluenesulfonyl or naphthylsulfonyl. Silyl means trimethylsilyl, triethylsilyl or tributylsilyl.

啉、二乙基胺、二環己基胺、普羅卡因、二苄基胺、N-苄基-β-苯乙基胺、1-非那明(1-phenamine)及N,N'-二苄基乙二胺等之含氮有機鹼的鹽等。就較佳鹽而言，可列舉藥理學上可容許的鹽，與鈉的鹽為更佳。As a salt of the compound of general formula [1], the salt in a generally known hydroxyl group is mentioned. For example, salts with alkali metals such as sodium and potassium; salts with alkaline earth metals such as calcium and magnesium; ammonium salts; and with trimethylamine, triethylamine, tributylamine, N-methylpiperidine pyridine, N-methyl

phenamine, diethylamine, dicyclohexylamine, procaine, dibenzylamine, N-benzyl-β-phenethylamine, 1-phenamine and N,N'-dibenzylamine Salts of nitrogen-containing organic bases such as benzylethylenediamine, etc. As a preferable salt, a pharmacologically acceptable salt is mentioned, and a salt with sodium is more preferable.

In the compound of the general formula [1], it is preferable that R ¹ is a hydrogen atom; R ² is a fluorine atom; and R ³ is a hydrogen atom. Moreover, the preferable compound is T-705.

Alternatively, in the compound of the general formula [1], it is preferable that R ¹ is a hydrogen atom; R ² is a hydrogen atom; and R ³ is a hydrogen atom. A preferred such compound is T-1105.

The compound of the general formula [1] is produced by combining well-known methods per se, for example, it can be produced by the production method described in the pamphlet of International Publication No. 00/10569.

衍生物或其鹽，例如T-705，係已知於細胞內遭受核糖基磷酸化，其結果所生成的吡

衍生物核糖三磷酸體會顯示抗病毒作用(Antimicrob Agents Chemother. 2005；49(3)：981-6.)。此處，吡

衍生物核糖三磷酸體係指通式[4]所表示的化合物

(式中，R¹ 及R² 係相同或相異而表示氫原子或鹵素原子；R³ 表示氫原子或胺基保護基)。Pyridine represented by the general formula [1]

Derivatives or salts thereof, such as T-705, are known to undergo ribosyl phosphorylation in cells, resulting in the formation of pyridoxine.

Derivatives ribose triphosphates show antiviral effects (Antimicrob Agents Chemother. 2005;49(3):981-6.). Here, pyridine

The derivative ribose triphosphate system refers to the compound represented by the general formula [4]

(In the formula, R ¹ and R ² are the same or different and represent a hydrogen atom or a halogen atom; R ³ represents a hydrogen atom or an amino protecting group).

In the compound of the general formula [2], it is preferable that R ⁴ , R ⁵ , R ⁶ and R ⁷ are hydrogen atoms; R ⁸ is methyl group; and X is oxygen atom. Also, the preferred compound is 6-methylmercaptopurine riboside.

In the combination of the compound of the general formula [1] and the compound of the general formula [2], the combination of T-705 and 6-methylmercaptopurine riboside is preferred.

衍生物或其鹽組合而使用之際，可增強通式[1]所表示的吡

衍生物或其鹽之抗RNA病毒的抗病毒活性。The thiopurine derivative represented by the general formula [2] is derived from a pyridine derivative represented by the general formula [1]

When used in combination with derivatives or their salts, the pyridine represented by the general formula [1] can be enhanced.

Antiviral activity of derivatives or salts thereof against RNA viruses.

衍生物或其鹽組合而使用之際，通過將細胞內的酵素或代謝路徑加以活性化或抑制等，而作為結果，使細胞內的吡

衍生物核糖三磷酸體的量增加。與未組合的情形進行比較，較佳為使吡

衍生物核糖三磷酸體之濃度增加1.5倍以上，更佳為增加1.9倍以上。前述抗病毒活性之增強作用之至少一部分係由於細胞內的吡

衍生物核糖三磷酸體之量增加所致。The thiopurine derivative represented by the general formula [2] is derived from a pyridine derivative represented by the general formula [1]

When the derivatives or their salts are used in combination, by activating or inhibiting enzymes or metabolic pathways in the cells, as a result, the pyridoxine in the cells is

Derivatives have increased amounts of ribose triphosphates. Compared with the uncombined situation, it is preferable to make the pyridine

The concentration of the derivative ribose triphosphate is increased by more than 1.5 times, more preferably by more than 1.9 times. At least part of the aforementioned enhancement of antiviral activity is due to intracellular pyridoxine

Derivatives due to the increase in the amount of ribose triphosphates.

The thiopurine derivative can be produced by combining known methods, or a commercially available one can be used. For example, 6-methylmercaptopurine riboside can be purchased from Fujifilm Wako Pure Chemical Industries, Ltd. and the like.

衍生物、與硫嘌呤衍生物而使用。組合係包含將吡

衍生物、與吡

衍生物同時地、各別地或以特定之順序投予的形態(併用)及作為混合物(摻合劑)的形態。In the present invention, the combination of pyridine

Derivatives, and thiopurine derivatives are used. Combination system contains pyridoxine

derivatives, and pyridine

The form in which the derivatives are administered simultaneously, separately or in a specific order (combination) and in the form of a mixture (admixture).

衍生物或其鹽以及硫嘌呤衍生物之投予時期相同者，「併用」亦包含：於1個之投予日程中投予吡

衍生物或其鹽以及硫嘌呤衍生物的形態。吡

衍生物或其鹽以及硫嘌呤衍生物之投予路徑可相同，亦可相異。That is, it does not mean only pyridine

Derivatives or their salts and thiopurine derivatives are administered at the same time, and "combination" also includes: administering pyridine in one administration schedule

Forms of derivatives or salts thereof and thiopurine derivatives. pyridine

The administration route of the derivatives or their salts and the thiopurine derivatives may be the same or different.

衍生物或其鹽、與硫嘌呤衍生物之量比，若為使吡

衍生物或其鹽之抗病毒活性增強的量比即可。較佳為吡

衍生物或其鹽：硫嘌呤衍生物(莫耳比)為1：500～500：1，更佳為1：200～200：1，進一步較佳為1：50～50：1，更較佳為1：10～10：1。pyridine

The ratio of derivatives or salts thereof to thiopurine derivatives, if pyridine

The amount ratio of the derivatives or their salts to enhance the antiviral activity is sufficient. preferably pyridine

Derivative or salt thereof: thiopurine derivative (mol ratio) is 1:500-500:1, more preferably 1:200-200:1, still more preferably 1:50-50:1, more preferably It is 1:10～10:1.

衍生物或其鹽以及硫嘌呤衍生物的情形，通常，可適當混合製劑化所使用的賦形劑、載劑及稀釋劑等之製劑輔助劑，此等可依據通常方法而作成錠劑、膠囊劑、散劑、糖漿劑、顆粒劑、丸劑、懸浮劑、乳劑、液劑、粉體製劑、栓劑、點眼劑、點鼻劑、點耳劑、貼附劑、軟膏劑或注射劑等之形態。Use the pyridine of the present invention

In the case of derivatives or their salts and thiopurine derivatives, usually, formulation auxiliary such as excipients, carriers, and diluents used for formulation can be appropriately mixed, and these can be made into tablets or capsules according to ordinary methods. Forms such as preparations, powders, syrups, granules, pills, suspensions, emulsions, liquids, powder preparations, suppositories, eye drops, nasal drops, ear drops, patches, ointments or injections.

衍生物或其鹽以及硫嘌呤衍生物在上述製劑化的過程中混合且均一化後，作成適當製劑即可。When used as an admixture, if the pyridine

The derivative or its salt and the thiopurine derivative may be mixed and homogenized in the above-mentioned formulation process, and then an appropriate formulation may be prepared.

衍生物或其鹽，可與硫嘌呤衍生物同時或以特定順序投予。The administration route of the therapeutic agent for RNA virus infection of the present invention is not particularly limited, and it can be administered by intravenous, oral, intramuscular, subcutaneous, inhalation, spray or other administration routes. Again, pyridine

The derivatives, or salts thereof, can be administered simultaneously or in a specific order with the thiopurine derivatives.

衍生物或其鹽之量，將0.1～1000mg/kg，較佳為0.1～100mg/kg，以1日1次至分割成數次進行投予即可。The administration method, dose, and frequency of administration can be appropriately selected according to the age, weight, and symptoms of the patient. Usually, in adults, when administered orally or parenterally (for example, by injection, drip, and administration to the rectal site, etc.), pyridoxine as an active ingredient

The amount of the derivative or its salt may be 0.1 to 1000 mg/kg, preferably 0.1 to 100 mg/kg, and it may be administered once a day to divided into several times.

The therapeutic agent for RNA virus infection of the present invention is useful for treatments such as treatment or prevention of RNA virus infection.

RNA virus infection refers to influenza virus, parainfluenza virus, Bunyavirus (Crimean Congo hemorrhagic fever virus), Rift Valley fever, Lak Lacrosse encephalitis virus, Dobrava virus, Maporal virus, Prospect hill virus, Andes virus, Sand fly fever virus ( sandfly fever virus), Heartland virus, Punta Toro virus, severe fever with thrombocytopenia syndrome virus, etc.), Arenavirus (Hu Junin virus, Pichinde virus, Tacaribe virus, Guanarito virus, Machupo virus, lymphocytic choriomeningeal inflammatory virus (lymphocytic choriomeningitis virus, Lassa fever virus, etc.), Filovirus (Ebola virus, Marburg virus, etc.), rabies virus, human interstitial pneumonia virus (Human metapneumovirus), RS virus, Nipah virus, Hendra virus, Measles virus, Hepatitis A virus, Hepatitis C virus, Hepatitis E virus, Chikungunya virus, Western Western equine encephalitis virus, Venezuelan encephalitis virus, Eastern equine encephalitis virus, Norovirus, Poliovirus, Human ECHO virus, Coxsackie virus, Enterovirus, Rhinovirus , Rotavirus, Newcastle disease virus, Mumps virus, Vesicular stomatitis virus, Japanese encephalitis virus, Tick-borne flavivirus, Yellow fever virus, dengue virus, West Nile virus, Zika fever virus or coronavirus (SARS coronavirus, SARS coronavirus-2, MERS coronavirus, new coronavirus (SARS- Infectious diseases caused by RNA viruses such as CoV-2 and its mutant viruses)). The present invention can preferably be used as influenza virus, rabies virus, Lassa fever virus, new bunya virus (Crimea Congo hemorrhagic fever virus, Rift Valley fever, severe febrile thrombocytopenia syndrome virus, etc.) and filovirus ( Ebola virus, Marburg virus, etc.) can be used as a therapeutic agent for infectious diseases, and it can be used as a therapeutic agent for influenza infection.

The therapeutic agent for RNA virus infection of the present invention may be used in combination with another therapeutic agent for RNA virus infection or a drug exhibiting an anti-RNA virus inhibitory effect for the purpose of enhancing the action or the like. The other therapeutic agent for RNA virus infection refers to a therapeutic agent for RNA virus infection, and examples thereof include a therapeutic agent for influenza, a therapeutic agent for hepatitis C, and a therapeutic agent for filovirus infection. Influenza therapeutic agents include amantadine, rimantadine, oseltamivir, zanamivir, peramivir, lanimir Wei (Laninamivir), Baloxavir (Baloxavir) and so on. Examples of therapeutic agents for hepatitis C include ribavirin, pegylated interferon, Telaprevir, Boceprevir, and galidevir. (Galidesivir) et al. The therapeutic agent for filovirus infection includes ribavirin, palivizumab, motavizumab, RSV-IGIV, MEDI-557, A-60444, MDT- 637, BMS-433771, amiodarone, dronedarone, verapamil, Ebola Convalescent Plasma, TKM-100201, BCX4430, FGI-106, TKM-Ebola, ZMapp, rNAPc2, OS-2966, MVA-BN filo, Brincidofovir, Ad26-ZEBOV, etc. Examples of agents exhibiting an anti-RNA virus inhibitory effect include mycophenolic acid, Daptomycin, Niclosamide, azithromycin, novobiocin, Chloroquine, memantine, prochlorperazine, chlorcyclizine, manidipine, Remdesivir, Imatinib, chlorpromazine ), nitazoxanide et al. (Journal of Young Pharmacists. 2019; 11(2): 117-121.). [Example]

Next, the present invention will be described with reference to examples, but the present invention is not limited to these examples.

衍生物與硫嘌呤衍生物之併用效果，以病毒的細胞感染模型進行試驗。Test Example 1 For pyridine

The effect of the combined use of the derivative and the thiopurine derivative was tested in a virus cell infection model.

衍生物。選擇6-甲基巰基嘌呤核糖苷作為硫嘌呤衍生物。選擇流感病毒作為RNA病毒。Select T-705 as pyridine

derivative. 6-Methylmercaptopurine riboside was chosen as the thiopurine derivative. Influenza virus was selected as the RNA virus.

(1) In the culture medium of Vero cells, in Eagle MEM/kanamycin 60 μg/mL (Iger MEM/kanamycin) medium supplemented with 10% fetal bovine serum, under the condition of 5% carbon dioxide, African green monkey kidney Vero cells subcultured at 37°C were detached by EDTA trypsin method, and a suspension containing 2×10 ⁴ cells in 100 μL prepared with the same medium was inoculated on 96-well plate. Under the condition of 5% carbon dioxide, the cells were cultured at 37°C overnight to obtain Vero cells that became a monolayer.

(2) Influenza virus infection and drug addition As a test medium, L-1-toluenesulfonamide-2-phenylethyl chloromethyl ketone (TPCK) treated with trypsin was added to Eagle MEM/conmycin medium at 1 μg/mL. culture medium. The culture supernatant of the Vero cells obtained in (1) was removed, and the following (A) to (C) were added to each well. After the drug was added, the cells were cultured at 35°C for 2 days under the condition of 5% carbon dioxide.

(A) 100 μL of Eagle’s MEM/Kanamycin medium (B) 50 μL of trypsin-treated Eagle’s MEM/Kanamycin medium containing 4 times the concentration of TPCK in the test medium was adjusted to 4.0×10 ³ PFU/ mL of influenza virus (PR/8/34(H1N1)) solution (C) 50 μL containing 4 times the set concentration of T-705 and 6-methylmercaptopurine riboside in combination of each set concentration containing 1% Eagle MEM/Kanamycin medium T-705 in DMSO set concentration (μM): 0, 0.1, 0.3, 1, 3, 10, 30, 100, 300, 1000 6-Methylmercaptopurine riboside set concentration ( μM): 0, 0.1, 0.3, 1, 3, 10

(3) Determination of cell degeneration effect (CPE) The CPE observed with the proliferation of influenza virus was determined in the following manner.

After the incubation, 50 μL of 100% formalin solution was added to each well to inactivate the virus and fix the cells. After standing at room temperature for more than 2 hours, the aqueous solution was removed and lightly washed with water, 50 μL/well of 0.02% methylene blue solution was added, and the solution was allowed to stand at room temperature for 1 hour. Remove 0.02% methylene blue solution, wash gently with water, and air dry. After that, absorbance (660 nm) was measured with a micropan analyzer (manufactured by Tecan, infinit M200). In the non-infected control group, 50 µL of trypsin-treated Eagle's MEM/conmycin medium containing TPCK at a concentration four times that of the test medium was used instead of the influenza virus solution, and the same operation as the test group was performed, and the absorbance was measured.

The test was carried out with 1 case per plate and 2 plates (the infected control group and the non-infected control group, for example, the number 8). A value obtained by subtracting the absorbance of the infected control group from the absorbance of the non-infected control group was defined as the value of complete inhibition of virus growth, and the CPE inhibition rate of each test was calculated from the following formula.

CPE inhibition rate=100×[(absorbance of single dose and combined use)-(absorbance of infection control group)]/[(absorbance of non-infected control group)-(absorbance of infection control group)]

The 50% CPE inhibitory concentration was calculated using the FORECAST function (first-order regression method) of Microsoft Office Excel 2010.

Table 1 shows the change in the 50% CPE inhibitory concentration of T-705 when 6-methylmercaptopurine riboside was added. Compared with the single dose of T-705, the 50% CPE inhibitory concentration of T-705 was lower in combination with 6-methylmercaptopurine riboside. In this test system, a single dose of 6-methylmercaptopurine riboside (10 μM) did not show any inhibitory effect on CPE. [Table 1] Changes in T-705 50% CPE inhibitory concentration due to addition of 6-methylmercaptopurine riboside 6-Methylmercaptopurine Riboside Concentration (µM) 0 0.1 0.3 1 3 10 T-705 50% CPE inhibitory concentration (µM) 183 89.4 48.9 30.6 21.9 20.2

Compared with the case of a single dose of T-705, in a cell infection model, it was confirmed that the combination of T-705 and 6-methylmercaptopurine riboside enhanced the antiviral activity.

衍生物或其鹽於細胞內受到核糖基磷酸化。例如已知T-705藉由核糖基磷酸化而生成的T-705-4-呋喃核糖基-5-三磷酸(T-705-4-ribofuranosyl-5-triphosphate(T-705RTP))，藉由抑制病毒的RdRp蛋白質而顯示抗病毒作用。為了確認於試驗例1所見的6-甲基巰基嘌呤核糖苷所致的T-705之活性增強效果是否是由於T-705 RTP的量的變化，而測定於Vero細胞中的6-甲基巰基嘌呤核糖苷處理下的T-705RTP量。Test Example 2 As mentioned above, pyridine

Derivatives or salts thereof undergo ribosyl phosphorylation in cells. For example, T-705-4-ribofuranosyl-5-triphosphate (T-705RTP) is known to be generated by ribosyl phosphorylation of T-705 by It inhibits the RdRp protein of the virus and exhibits antiviral effect. In order to confirm whether the activity-enhancing effect of T-705 by 6-methylmercaptopurine riboside seen in Test Example 1 is due to the change in the amount of T-705 RTP, the 6-methylmercapto riboside in Vero cells was measured. Amount of T-705RTP under purine riboside treatment.

(1) Determination of the amount of T-705RTP treated with 6-methylmercaptopurine riboside in Vero cells (1-1) Extraction of intracellular T-705RTP 2×10 ⁵ cells were inoculated into each well of a 6-well plate Cells of Vero cells were cultured in vehicle (0.0125% DMSO) or various concentrations of 6-methylmercaptopurine riboside, 100 μM T-705, or both agents, at 37°C under 5% carbon dioxide for 24 days. hours (case number 3).

After culturing, after washing with PBS, 300 μL of trypsin was put in, and the cells were detached by standing at 37° C. for 5 minutes. 700 μL of Eagle’s MEM medium supplemented with 10% fetal bovine serum was added to suspend, and collected in a 1.5 mL vial. After that, 10 μL was used for cell number measurement. The entire remaining cell suspension was centrifuged at 1,000 rpm and 4°C for 5 minutes (MX-207, TOMY), and the supernatant was removed. Next, 1 mL of PBS was added to the cell pellet for resuspension, and after centrifugation at 1,000 rpm and 4°C for 5 minutes again, the supernatant was removed. After that, 300 μL of 70% methanol was added to suspend well. The samples were then assayed using a high-speed liquid chromatograph (HPLC-FL) with a fluorescence detector.

甲醯胺(6-bromo-3-hydroxy-2-pyrazinecarboxamide)溶液20μL及蒸餾水180μL，將離心分離(約1600rpm，室溫，5分鐘，MICRO SINKER)後的上清液以HPLC進行分析。於HPLC分析使用C18管柱，藉由於弱酸性移動相中添加離子對試藥(四丁基溴化銨)，而使T-705RTP保持於管柱中。螢光檢出的激發波長為370nm，螢光波長設為445nm。由各細胞樣品之HPLC面積比(T-705RTP/內標準)，藉由校正線的逆回歸而求出T-705RTP濃度。將T-705RTP量的變化示於表2。得知藉由6-甲基巰基嘌呤核糖苷，T-705處理後之細胞內T-705RTP量增加。 [表2] Vero細胞內T-705RTP量的變化 6-甲基巰基嘌呤核糖苷濃度 (µM) 0 1 10 T-705RTP濃度 (pmol/10⁶ 個細胞) 88.3 148 167 (1-2) Measurement of cell number and HPLC-FL analysis The cell number measurement and HPLC-FL analysis were performed to determine the amount of T-705RTP in each sample. Add 6-bromo-3-hydroxy-2-pyridine as an internal standard to 50 μL of the well-stirred cell sample (prepared in (1-1) above) under ice-cooling

20 μL of carboxamide (6-bromo-3-hydroxy-2-pyrazinecarboxamide) solution and 180 μL of distilled water, and the supernatant after centrifugation (about 1600 rpm, room temperature, 5 minutes, MICRO SINKER) was analyzed by HPLC. A C18 column was used for HPLC analysis, and T-705RTP was kept in the column by adding an ion pair reagent (tetrabutylammonium bromide) to the weakly acidic mobile phase. The excitation wavelength for fluorescence detection was 370 nm, and the fluorescence wavelength was 445 nm. From the HPLC area ratio (T-705RTP/internal standard) of each cell sample, the T-705RTP concentration was determined by inverse regression of the calibration line. Table 2 shows the change in the amount of T-705RTP. It was found that the amount of intracellular T-705RTP increased after T-705 treatment by 6-methylmercaptopurine riboside. [Table 2] Changes in the amount of T-705RTP in Vero cells 6-Methylmercaptopurine Riboside Concentration (µM) 0 1 10 T-705RTP concentration (pmol/ ¹⁰ cells) 88.3 148 167

The mechanism of action of T-705, that is, it exhibits antiviral activity after being converted into T-705RTP in cells, is applicable regardless of the type of virus as long as it is a virus for which T-705 has an effect. Since the amount of T-705 RTP in cells is increased by the thiopurine derivative, it can be said that the combination of T-705 and the thiopurine derivative simultaneously enhances the antiviral activity of a plurality of RNA viruses that have an effect on T-705.

衍生物或其鹽。例如，除了T-705以外，於通式[1]之化合物中，R¹ 為氫原子；R² 為氫原子；R³ 為氫原子的化合物(3-羥基-吡

甲醯胺)顯示抗病毒活性(Antiviral Res. 2009；82(3)：95-102.)，暗示著於細胞內變換為核糖三磷酸體(Mol Pharmacol.2013；84(4)：615-29.)。即，可藉由組合吡

衍生物或其鹽、與硫嘌呤衍生物，而同時增強對吡

衍生物或其鹽具有效果的複數之RNA病毒的抗病毒活性。 [產業上利用之可能性]This mechanism is not only applicable to T-705, but also widely applicable to the pyridine represented by the general formula [1].

derivatives or their salts. For example, except for T-705, in the compounds of the general formula [1], R ¹ is a hydrogen atom; R ² is a hydrogen atom; R ³ is a hydrogen atom (3-hydroxy-pyridine

formamide) showed antiviral activity (Antiviral Res. 2009; 82(3): 95-102.), suggesting intracellular conversion to ribose triphosphates (Mol Pharmacol. 2013; 84(4): 615-29 .).) That is, by combining pyridine

Derivatives or their salts, and thiopurine derivatives, while enhancing the

Derivatives or salts thereof have an effective antiviral activity against a plurality of RNA viruses. [Possibility of Industrial Use]

衍生物或其鹽、與硫嘌呤衍生物而構成的RNA病毒感染症治療劑，顯示增強的抗病毒活性，於醫藥產業的領域為有用的。combination pyridine

A therapeutic agent for RNA virus infection composed of a derivative or a salt thereof and a thiopurine derivative exhibits enhanced antiviral activity and is useful in the field of the pharmaceutical industry.

without.

without.

without.

Claims (4)

A therapeutic agent for RNA virus infection, which is a combination of pyridoxine represented by general formula [1]

A therapeutic agent for RNA virus infection composed of a derivative or a salt thereof, and a thiopurine derivative represented by the general formula [2],

(in the formula, R ¹ and R ² are the same or different and represent a hydrogen atom or a halogen atom; R ³ represents a hydrogen atom or an amino protecting group),

(in the formula, R ⁴ and R ⁵ are the same or different and represent a hydrogen atom or a hydroxyl protective group; R ⁶ represents a hydrogen atom, a monophosphate group that can be protected, a diphosphate group that can be protected or a protective group that can be protected Triphosphoric acid group; R ⁷ represents a hydrogen atom or a protectable amine group; R ⁸ represents a hydrogen atom, a C _1-6 alkyl group or a group represented by the general formula [3]

(in the formula, R ⁹ represents a hydrogen atom, a C _1-6 alkyl group or a carboxyl group; R ¹⁰ represents a hydrogen atom, a C _1-6 alkyl group, a benzyl group or a p-nitrobenzyl group); X represents an oxygen atom , a sulfur atom, a carbon atom or a protectable imino group). The therapeutic agent of claim 1, wherein R ¹ is a hydrogen atom, R ² is a fluorine atom or a hydrogen atom, and R ³ is a hydrogen atom. The therapeutic agent of claim 1 or 2, wherein R ¹ is a hydrogen atom, R ² is a fluorine atom, and R ³ is a hydrogen atom. The therapeutic agent of claim 1 or 2, wherein R ⁴ , R ⁵ , R ⁶ and R ⁷ are hydrogen atoms, R ⁸ is a methyl group, and X is an oxygen atom.